The focus of experiments in Phase I will be on setting up systems and developing techniques that will be used in Phase II to define mRNA structural features which mediate nucleocytoplasmic transport selectivity nd efficiency. Two living cell systems will be sued: Xenopus oocytes and cultured Cos-I cells. In Phase I, the normal transport pattern will be determined for mRNAs transcribed rom the alpha-globin gene and an alpha-globin chimeric minigene in micro-injected oocytes and transfected Cos-1 cells. After expression levels adequate for assay are demonstrated in both systems, relevant baseline data, such as nuclear mRNA stability, cytoplasmic mRNA stability, and rate of cytoplasmic mRNA accumulation, will be determine by a variety of mRNA analytic approaches. Based on Phase I control experiments, Phase II studies will address the effect of site-specific secondary structure on transport. Alpha-globin cDNA fragments of defined size and extent isolated from a Bal31 digest library will be inserted into the 3'NTR of the alpha-globin gene and an alpha-globin minigene. These recombinant genes, which are predicted to encode mRNAs with defined, extensive, and highly stable secondary structures, will be injected is not oocyte nuclei or transfected into Cos-1 cells and cytoplasmic mRNA levels assayed. Nucleocytoplasmic transport of these mRNAs, which contain site-specific secondary structures, can be compared to the expression of control mRNAs that lack such structures (determined in Phase I) but are otherwise identical. In this manner, the site-specific effects of secondary structure on nucleocytoplasmic transport can be assayed, and the structural features that determine transport specificity and efficiency can begin to be determined.